The chemiluminescent reaction is that between luminol, a peroxide source and a peroxydase enzyme, especially horseradish peroxidase (HRP), that catalyzes the oxidation of luminol by peroxide. Such oxidation is accompanied by light emission.
The chemiluminescent oxidation of luminol catalyzed by peroxidase finds wide employment in analytical tests of antigens, antibodies and nucleic acids, and in particular in blotting tests, e.g., Dot Blots, Western Blots (proteins), Southern and the Northern Blots (nucleic acids).
It is known that the chemiluminescent oxidation of luminol catalyzed by peroxidase can be made faster and more efficient by adding an electron mediator, or enhancer, as shown, for example, by L. J. Kricka in Clinical Chemistry 1991; 37:1472-1481; or by L. J. Kricka, J. C. Voyta and I. Bronstein in “Chemiluminescent Methods for Detecting and Quantitating Enzyme Activity”, Methods Enzymol. 2000; 305:370-390. Several compounds have been used as electron mediators. In particular, firefly luciferin, 6-hydroxybenzotriazole, p-iodophenol, p-coumaric acid are described by G. H. G. Thorpe and L. J. Kricka, Methods Enzymol. 1986; 133:331; aromatic amines in U.S. Pat. No. 4,279,950; acetanilides in Eur. Pat. Appl. No. 603953 (1994); the indophenols and phenothiazines N-substituted and indophenols in U.S. Pat. No. 5,171,668; boronic acids replaced in U.S. Pat. No. 5,629,168. It is believed that in the presence of an electron mediator, the oxidation of luminol catalyzed by peroxidase proceeds as follows:HRP+H2O2→HRP-I  (1)HRP-I+LH−→HRP-II+L•−  (2)HRP-II+LH−→HRP+L•−  (3)HRP-I+E→HRP-II+E•  (4)HRP-II+E→HRP+E•  (5)E•+LH−→E+L•−  (6)L•−→L+LH−  (7)L+H2O2→LO22−  (8)LO22−→(AP2−)*+N2  (9)(AP2−)*→AP2−+hν  (10)where HRP, HRP-I and HRP-II indicate the enzyme peroxidase in the native form and in its two oxidized forms, respectively; LH−, LH•−, L, LO22− represent luminol anion, luminol radical anion, diazaquinone and luminol peroxide; E ed E• represent the electron mediator and its corresponding radical; finally, AP2− indicates the dianion of 3-aminophthalic acid, and (AP2−)* its excited state. According to this scheme, peroxidase HRP is oxidized by peroxide to HRP-I. The luminol anion and the enhancer are oxidized by HRP-I to their respective radicals with conversion of the enzyme to its HRP-II form. In turn, HRP-II oxidizes another molecule of luminol anion or of electron mediator to their respective radicals, simultaneously regenerating the native form of the HRP enzyme, which can participate in another oxidation cycle. It is thus believed that the increase in the chemiluminescent signal is due to the faster generation of the key intermediate LH•− in the presence of an electron mediator (see e.g., S. B. Vlasenko, A. A. Arefyev, A. D. Klimov, B. B. Kim, E. L. Gorovits, A. P. Osipov, E. M. Gavrilova, A. M. Yegorov, J. Biolumin. Chemilumin. 1989; 4:164-176, or B. Cercek, K. Roby, L. Cercek, J. Biolumin. Chemilumin. 1994; 9:273-277).
The subsequent phases of the chemiluminescent reaction are less clear. The radical anion of luminol LH•− is unstable and can dismutate to luminol anion LH− and diazaquinone, L. The diazaquinone in turn is likely to be susceptible to nucleophilic attack by peroxide ion HO2− on the carbonylic carbon (C═O), with formation of luminol peroxide LO22−, in the open or cyclical form (endoperoxide). Finally, luminol peroxide collapses to 3-aminophthalate, AP2−, with expulsion of molecular nitrogen. Some of the energy thus produced is captured by aminophthalate with formation of its excited state (AP2−)* and subsequent emission of blue light (425 nm). The efficiency of this process corresponds to the fluorescence quantum yield of 3-aminophthalate (approximately 30%). Although the exact details of reactions (7)-(9) are not known, it is conceivable that the conversion of luminol radical anion, LH•−, to luminol peroxide, LO22−, involves nucleophilic attack of peroxide ion on a carbonylic carbon (C═O) of luminol (see e.g., G. Merenyi, J. Lind and T. E. Eriksen “Nucleophilic Addition to Diazaquinones. Formation and Breakdown of Tetrahedral Intermediates in relation to Luminol Chemiluminescence,” J. Am. Chem. Soc. 1986; 108:7716-7726.) On the other hand, the efficiency of this reaction is decisive for the formation of (AP2−)*.